Iron is essential for cell growth, proliferation and differentiation. Heme-iron is a cofactor for hemoglobin and myoglobin involved in many important physiological processes including oxygen binding and transport, and oxygen metabolism. Non-heme iron is the active center of many important enzymes involved in DNA synthesis and cell cycle (Pantopoulos, K. et al, Regulation of cellular iron metabolism. Biochem. J. 2011, 434, 365-381). Iron is also a necessary cofactor for the synthesis of neurotransmitters, dopamine, norepinephrine, and serotonin, and disruption of iron homeostasis may be involved in Parkinson's disease and/or mood disorders (Youdim, 1990).
Iron deficiency is the most common cause of anemia and represents a global health problem. Iron-deficiency anemia is defined by low numbers of small (microcytic) and hypoferremic erythrocytes. Iron deficiency may contribute to cognitive developmental defects in children, poor physical performance, and unfavorable pregnancy outcomes (Camaschella, 2015). Iron deficiency in children results in auditory defects from disruption of myelin (Roncagliolo et al., 1998), and demylinating diseases such as multiple sclerosis are associated with defects in cellular iron homeostasis (Drayer et al., 1987).
Iron overload is also common and equally detrimental, affecting parenchymal organs including the liver, heart, and pancreas. In Western populations iron overload is mostly genetic due to hereditary hemochromatosis (HH), caused by mutations in genes involved in the sensing of systemic iron levels (such as HFE, HJV, and TFR2), or to disorders that cause ineffective erythropoiesis and secondary iron loading (e.g., thalassemias). There is increasing awareness that acquired metabolic disorders can also cause iron overload, which may exacerbate pathogenesis (Pietrangelo, 2016).
In the retina, iron is particularly important for the visual phototransduction cascade. Photoreceptor cells are constantly shedding and synthesizing their outer segments containing disc membranes. Thus, photoreceptors depend highly on iron-containing enzymes including fatty acid desaturase for synthesis of lipids used in generating new disc membranes (Schichi, 1969).
While iron is necessary for retinal function, excess iron can be harmful. Free Fe2+ participates in the Fenton reaction by catalyzing the conversion of hydrogen peroxide to the hydroxyl radical, the most reactive of reactive oxygen species. Hydroxyl radicals are extremely reactive, causing lipid peroxidation, DNA strand breaks, and degradation of biomolecules (Halliwell and Gutteridge, 1984), and have been implicated in the pathogenesis of Alzheimer's and other CNS diseases (Smith et al., 1997).
The retina is isolated from the bloodstream by blood-retinal barriers. The retinal pigment epithelium (RPE) and the neuroretinal vasculature form independent barriers, the intercellular tight junctions of which prevent intercellular diffusion, thereby protecting both sides of the retina from the systemic circulation.
Ferric iron is carried in the bloodstream in association with a protein, transferrin (Baker and Morgan, 1994). Transferrin, with iron, is endocytosed into cells following binding to the cell surface transferrin receptor. The transferrin is found in the retina (Yefimova et al., 2000). Transferrin mRNA expression was detected by in situ hybridization in the RPE cell layer, indicating that the RPE is the main site of transferrin synthesis. Transferrin may carry iron from the RPE to the photoreceptors via a Tf-TfR-dependent mechanism (Yefimova et al., 2000).
Iron bound to transferrin in the choroidal circulation has been shown to be taken up by high-affinity transferrin receptors at the basolateral surfaces of RPE cells. From there, iron is transported to the apical surfaces of RPE cells where it is released to the neural retina.
Increased intraocular iron has been found to cause oxidative damage to the retina. A higher concentration of iron causes the outer border of the outer nuclear layer to become irregular, suggesting photoreceptor damage. People with a deficiency in ceruloplasmin resulting from the recessive disease aceruloplasminemia also have retinal iron accumulation with retinal degeneration.
Disruption in iron homeostasis between the retina and RPE may also cause iron overload. Non-heme iron was found to build up in this debris layer in a time-dependent manner with photoreceptor degeneration, while transferrin levels in the photoreceptor layer were diminished. Photoreceptor loss starts at postnatal day 20 and is significantly increased one month later. In this model, the disruption of normal RPE-photoreceptor interactions leads to an iron homeostasis disorder, which may ultimately contribute to retinal degeneration.
Recent studies suggest that abnormal retinal iron metabolism may promote a variety of retinal disorders. These include ocular siderosis either from intraocular foreign bodies or from intraocular hemorrhage.
Retinal degeneration has also been observed in hereditary disorders resulting in iron overload, including aceruloplasminemia, hereditary hemochromatosis, pantothenate kinase associated neurodegeneration (formerly Hallervorden-Spatz Disease), and Friedreich's Ataxia.
Recently, evidence suggests that iron overload may also play a role in the pathogenesis of age-related macular degeneration (AMD) that leads to vision loss. Possible mechanisms of this vision loss include direct iron toxicity to the photoreceptors, iron toxicity or mechanical damage to the RPE, cellular migration and proliferation in the subretinal space, proliferation of fibrovascular membrane, or separation of photoreceptors from the RPE (Gillies and Lahav, 1983).
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed nations in people age 65 and older (Klein et al., 1995; Leibowitz et al., 1980). Iron may be a source of oxidants in AMD. AMD-affected maculas (n=10) had more iron than healthy age-matched maculas (n=9), as demonstrated by an enhanced Perls' Prussian blue stain on sections of the optic disk and macula followed by computer-assisted analysis of digital images to quantify the stain (Hahn et al., 2003).
As age-related macular degeneration may be caused by iron-mediated oxidative damage, it is assumed that antioxidants and iron chelators may be effective in reducing the occurrence and progression of AMD. While the Age-Related Eye Disease Study (AREDS) has shown that supplemental zinc, vitamin C, vitamin E, and β-carotene can provide a protective effect on AMD progression, it is likely that additional antioxidants may further prevent or slow the progression of AMD.
Since iron is one of the most potent generators of oxidative damage through production of hydroxyl radicals in the Fenton reaction, and since the antioxidants used in the AREDS study may not quench all hydroxyl radical produced by iron, it is possible that iron chelators will prove a useful adjunct to AREDS vitamins.
Recent researches suggest that iron chelation may play a role in the treatment of a number of neurological diseases such as Alzheimer's disease and Parkinson's disease, Huntington's disease and Friedreich's Ataxia (Zheng et al., 2005; Richardson, 2004). It is plausible that iron chelation may also be useful in retinal disease associated with iron overload.
Until recently, the only iron chelator in widespread clinical use in the United States was deferoxamine B (DFO), and despite being a relatively effective iron chelator for the treatment of transfusional iron overload, it has many notable limitations. The drug is an inefficient iron chelator, as only 5% or less of the drug administered promotes iron excretion (Bergeron et al., 2002).
In addition, because the iron chelator is poorly absorbed by the gastrointestinal system, and its elimination from the body is rapid, effective DFO treatment requires subcutaneous (SC) or IV administration for 9 to 12 hours for 5 or 6 days each week (Lee et al., 1993; Pippard, 1989). Therefore, for chronic treatment, chelation with DFO is costly, inefficient, cumbersome, and unpleasant.
In addition, DFO administration can have some rare but potentially serious side effects, including pulmonary toxicity, bony changes, growth failure, and promotion of Yersinia enterocolitica infections (Tenenbein et al., 1992; Brill et al., 1991; De Virgiliis et al., 1988).
Other iron chelators have been put into clinical use, including deferiprone (L1) and deferasirox (Exjade). Deferiprone has the advantage of being orally active and has been shown to be a more efficient iron chelator than DFO in removing cardiac iron, the cause of most of the mortality in transfusional iron overload (Anderson et al., 2002). A recent report demonstrates the ability of L1 to decrease brain iron in patients with Friedreich's Ataxia (Boddaert et al., 2007). This result suggests that L1 may similarly decrease retinal iron levels.
Deferiprone has rare but serious side-effects, including hepatic fibrosis, agranulocytosis, neutropenia, and arthropathy (Olivieri et al., 1986; Cohen et al., 2003; Ceci et al., 2002). The cause of deferiprone-related side effects is not known, but it may be deferiprone is a bidentate iron chelator.
At low concentrations, bidentate iron chelators can actually facilitate the formation of free-radicals from the formation of incomplete iron chelator complexes (Hershko et al., 2005). Since three molecules of deferiprone are required to completely remove iron from the labile pool, low levels of deferiprone can leave iron incompletely chelated and may cause the unbound portion of iron to be an even more effective catalyst for the generation of free radicals.
Deferasirox is an iron chelator that has just been recently approved for clinical use in patients with iron overload due to blood transfusion. Deferasirox is orally active and has an extended half-life, allowing for once-daily oral dosing (Vanorden and Hagemann, 2006). Current data show deferasirox to be as effective an iron chelator as subcutaneous deferoxamine, which is the current drug of choice for chronic iron overload patients (Piga, et al., 2002).
Another potentially therapeutic iron chelator with interesting properties is salicylaldehyde isonicotinyl hydrazone (SIH). This iron chelator can protect cultured cardiomyocytes from oxidative stress induced death at concentrations 1000 fold lower than DFO (Simunek et al., 2005). However, SIH has poor stability in an aqueous environment due to the rapid hydrolysis of its hydrazone bond.
There are many challenges with using these clinically-available iron chelators to prevent and treat retinal degeneration. Ideally, an iron chelator should be selectively bind iron, but not other biologically important divalent metals such as Zinc (Liu and Hider, 2002).
In addition, an effective iron chelator must reach its target sites at a sufficiently high level. The chelator must be able to be absorbed in sufficient quantity through the gastrointestinal tract, the blood-brain barrier, or in the case of the retina, the blood-retina barrier (BBB). Thus, to successfully penetrate the blood-brain/blood-retinal barrier, an iron chelator must possess appreciable lipid solubility (Kalinowski and Richardson, 2005) and small molecular size, ideally below 500 Daltons (Maxton et al., 1986).
Iron must be carefully regulated to provide necessary iron levels without causing oxidative damage in the photoreceptors, where there is a high oxygen tension and high concentration of easily oxidized polyunsaturated fatty acids,
Iron that is not utilized or stored by the cell may be exported by the transport protein ferroportin (also known as MTP-1 or IREG-1) (Donovan et al., 2000; Abboud and Haile, 2000; McKie et al., 2000). Iron is exported by ferroportin in its ferrous state and must be oxidized to be accepted by circulating transferrin.
The oxidation of ferrous iron is accomplished by ferroxidases, ceruloplasmin and hephaestin. Ceruloplasmin is a copper binding protein, which contains over 95% of copper found in plasma. Hephaestin has 50% homology to ceruloplasmin and has ferroxidase activity. Unlike ceruloplasmin, which is present as a secreted plasma protein and glycosylphosphatidylinositol (GPI)-anchored protein (Patel and David, 1997), hephaestin is present only as a membrane-bound protein.
The opposing requirements and toxicities of iron are managed by an iron-responsive mechanism of post-transcriptional regulation of key iron-handling proteins (Hentze and Kuhn, 1996). This regulation allows individual cells to regulate iron uptake, sequestration, and export according to their iron status.
Further, there is no known mechanism of iron excretion from the body. Roughly 1-2 mg of iron is lost daily through sweat, blood loss, sloughing of intestinal epithelial cells, and desquamation. To compensate for this loss, the body absorbs about 1-2 mg of dietary iron per day, but hemoglobin synthesis alone requires 20-25 mg of iron per day.
To support hemoglobin synthesis and other metabolic processes, iron must be recycled and tightly regulated within the system instead of chelation. The circulating peptide hormone hepcidin together with its receptor ferroportin primarily maintain systemic iron homeostasis, whereas iron-regulatory proteins play a primary role in the control of intracellular homeostasis (https://www.ncbi.nlm.nih.gov/pme/articles/PMC4464783/)
The management of iron levels and delivery is also a major challenge. Human cells accumulate iron from two main circulating sources. The first one, which is a classical source, consists of iron bound to transferrin, as described below, and the second one is called Non-Transferrin-Bound Iron (NTBI).
Most cell use transferrin, a serum protein, as a primary staple iron source/transporter. Transferrin comprises a class of biological iron-binding proteins, each lobe bearing a single site capable of reversibly binding iron and accounting for the physiological roles of the proteins in iron transport and iron withholding as a defense against infection.
Tf normally provides iron for cellular needs and for most cells, the delivery of transferrin-borne iron depends on association of the protein with transferrin receptors, TfR1 and TfR2, on plasma membranes. An elaborate receptor-mediated pathway drives endocytosis of Tf-bound iron into mammalian cells for use and storage. Thus, TfR1 and TfR2 play critical roles in iron transfer involving transferrin.
For iron deficient patients, an effective transport of iron from external sources into the cells is required. This requirement is complicated by the fact that environmental iron is invariably present as insoluble iron leading to poor bioavailability and toxicity. Therefore, activators which provide efficient uptake and transport systems to extract iron from their environment and ferritins that store iron in a non-toxic form are required.
Iron-regulatory proteins (IRPs) register intracellular iron status and, in cases of intracellular iron deficiency, bind to iron-responsive elements (IREs) on the mRNA of the regulated protein. Binding of IRPs to the IRE of ferritin sterically obstructs efficient translation, which decreases ferritin levels in iron-deficiency. In contrast, binding of IRP to the IRE of transferrin receptor protects mRNA from degradation, which increases transferrin receptor in iron-deficiency.
Therefore, there is a need for effective intracellular iron regulatory mechanism that can balance and regulate iron within retina cells.